Monitoring Astrocytic Proteome Dynamics by Cell Type-Specific Protein Labeling

• Published in: PloS one Vol. 10; no. 12; p. e0145451
• Main Authors: Müller, Anke, Stellmacher, Anne, Freitag, Christine E, Landgraf, Peter, Dieterich, Daniela C
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 21.12.2015; Public Library of Science (PLoS)
• Subjects: Alanine - analogs & derivatives; Alanine - chemistry; Amino acids; Analysis; Animals; Astrocytes; Astrocytes - cytology; Astrocytes - drug effects; Astrocytes - metabolism; Attention; Azides - analysis; Azides - chemistry; Brain; Brain research; Brain-derived neurotrophic factor; Brain-Derived Neurotrophic Factor - pharmacology; Cell culture; Coculture Techniques; Communication; Connexin 43; Dissociation; Gene expression; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; HEK293 Cells; Humans; Insects; Labeling; Labelling; Mass spectrometry; Metabolism; Methionine-tRNA Ligase - genetics; Methionine-tRNA Ligase - metabolism; Nervous system; Neurobiology; Neuroglia - cytology; Neuronal-glial interactions; Neurons; Neurosciences; Norleucine - analogs & derivatives; Norleucine - analysis; Norleucine - chemistry; Pharmacology; Plastic foam; Plastic properties; Plasticity; Protein Biosynthesis; Protein expression; Protein synthesis; Proteins; Proteins - chemistry; Proteins - metabolism; Proteome; Proteomes; Proteomics; Proteomics - methods; Rats, Wistar; Ribosomal protein L10a; Rodents; Scientific imaging; Synaptic plasticity; Synthesis; Tagging; Toxicology; Transfer RNA; tRNA; Germany
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0145451

The ability of the nervous system to undergo long-term plasticity is based on changes in cellular and synaptic proteomes. While many studies have explored dynamic alterations in neuronal proteomes during plasticity, there has been less attention paid to the astrocytic counterpart. Indeed, progress in identifying cell type-specific proteomes is limited owing to technical difficulties. Here, we present a cell type-specific metabolic tagging technique for a mammalian coculture model based on the bioorthogonal amino acid azidonorleucine and the mutated Mus musculus methionyl-tRNA synthetaseL274G enabling azidonorleucine introduction into de novo synthesized proteins. Azidonorleucine incorporation resulted in cell type-specific protein labeling and retained neuronal or astrocytic cell viability. Furthermore, we were able to label astrocytic de novo synthesized proteins and identified both Connexin-43 and 60S ribosomal protein L10a upregulated upon treatment with Brain-derived neurotrophic factor in astrocytes of a neuron-glia coculture. Taken together, we demonstrate the successful dissociation of astrocytic from neuronal proteomes by cell type-specific metabolic labeling offering new possibilities for the analyses of cell type-specific proteome dynamics.